Slowing Motor Nerve Conduction Velocity Streptozotocin ...TM In thepresent studyweexaminedthe...

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Slowing of Motor Nerve Conduction Velocityin Streptozotocin-induced Diabetic Rats is Precededby Impaired Vasodilation in Arterioles that Overliethe Sciatic NerveLAWRENCE J. COPPEY, ERIC P. DAVIDSON, JOYCE A. DUNLAP,DONALD D. LUND and MARK A. YOREK*

Veterans Affairs Medical Center, Diabetes Endocrinology Research Center and Department of Internal Medicine,University of Iowa, Iowa City, IA, 52246

(Received in final form 14 February 2000)

Diabetes mellitus produces marked abnormalities inmotor nerve conduction, but the mechanism is notclear. In the present study we hypothesized that inthe streptozotocin (STZ)-induced diabetic rat im-paired vasodilator function in arterioles that providecirculation to the region of the sciatic nerve isassociated with reduced endoneural blood flow(EBF) and that these defects precede slowing ofmotor nerve conduction velocity, and thereby maycontribute to nerve dysfunction. As early as threedays after the induction of diabetes endoneuralblood flow was reduced in the STZ-induced diabeticrat. Furthermore, after I week of diabetes acetylcho-line-induced vasodilation was found to be impaired.This was accompanied by an increase in the super-oxide level in arterioles that provide circulation tothe region of the sciatic nerve as well as changes inthe level of other markers of oxidative stressincluding an increase in serum levels of thiobarbi-turic acid reactive substances and a decrease in lensglutathione level. In contrast to the vascular relatedchanges that occur within I week of diabetes, motornerve conduction velocity and sciatic nerve Na//KATPase activity were significantly reduced follow-ing 2 and 4 weeks of diabetes, respectively. These

studies demonstrate that changes in vascular func-tion in the STZ-induced diabetic rat precede theslowing of motor nerve conduction velocity (MNCV)and are accompanied by an increase in superoxidelevels in arterioles that provide circulation to theregion of the sciatic nerve.

Keywords: Diabetes, vasodilation, diabetic neuropathy, acet-ylcholine, oxygen radicals, endothelium

INTRODUCTION

The purpose of this study was to examine therelationship between diabetes-induced reduc-tion in sciatic nerve motor nerve conductionvelocity, endoneural blood flow and vascularreactivity of arterioles that provide circulationto the region of the sciatic nerve using thestreptozotocin-induced diabetic rat model.

*Corresponding author. 3 E 17 Veterans Affairs Medical Center, Iowa City, IA 52246. Tel.: (319) 338-0581 ext. 7630, Fax: (319)339-7025, e-mail: [email protected]

131

132 L.J. COPPEY et al.

In a previous study we showed that acetylcho-line-induced endothelium-dependent vasodila-tion of arterioles that provide blood flow to theregion of the sciatic nerve is impaired bydiabetes. Lll This defect was associated with adecrease in neuronal blood flow of the sciaticnerve and slowing of motor nerve conductionvelocity. In the present study we investigatedwhether the impaired vasodilation of thesearterioles in diabetic rats preceded the changesin endoneural blood flow and nerve conduction.

Diabetes mellitus produces marked abnor-malities in motor nerve conduction, but themechanism is not clear. Cameron et al. reportedthat hyperglycemia-induced blood flow reduc-tion and resultant endoneural hypoxia are im-portant factors underlying nerve conductiondeficits early in the development of diabeticneuropathy. [2] In their studies, they showed thatnerve blood flow was reduced as early as I weekafter diabetes induction. In other studies con-ducted with older animals reductions in nerveblood flow were apparent and preceded thedecrease in nerve conduction velocities. TM Inthe present study we examined the relationshipbetween changes in endoneural blood flow andnerve conduction velocity induced by diabetes,and the impairment in acetylcholine-inducedvasodilation in arterioles that provide circula-tion to the region of the sciatic nerve.The effect of diabetes on vascular reactivity

has been studied in a variety of vascular tissues.In the aorta from several species, agonist-in-duced endothelium-dependent vasodilation isimpaired by diabetes and by acute hyperglyce-mia. [4-7] Similar alterations in vasodilator re-sponses have been observed in mesentericarteries from diabetic rats. I8-11 Other studieshave shown reduced acetylcholine-induced va-sodilation of the basilar artery, [12, 13] renal inter-lobar artery, [14] and coronary artery [15,16] indiabetes. Therefore, impaired endothelial-depen-dent vasodilation of the aorta and arteriessupplying the kidney, heart and as reportedrecently by us, the region of the sciatic nerve,

is a common defect in diabetes. In this study wereport that a reduction in endoneural blood flowin the sciatic nerve occurs as early as three daysafter the induction of diabetes. Acetylcholine-induced vasodilation by arterioles that providecirculation to the region of the sciatic nerve isalso impaired early in diabetes and is accom-panied by an increase in superoxide levels inthese vessels. These changes precede the slow-ing of motor nerve conduction velocity andthe reduction in Na+/K+ ATPase activity in thesciatic nerve.

METHODS

Animals

Male Sprague-Dawley (Harlan Sprague Daw-ley, Indianapolis, IN) rats 8-9 weeks of age wereused for these studies. The animals were housedin a certified animal care facility and food andwater were provided ad libitum. All institution-al and NIH guidelines for use of animals werefollowed. Diabetes was induced by intrave-nously injecting streptozotocin (60 mg kg-1 in0.9% NaC1, adjusted to a pH 4.0 with 0.2Msodium citrate). Control rats were injected withvehicle alone. The rats were anesthetized withmethoxyflurane before injection. Diabetes wasverified 24 h later by evaluating blood glucoselevels with the use of glucose oxidase reagentstrips (Boehringer- Mannheim, Indianapolis,IN). Rats having blood glucose level of 300mg/dl (16.7mM) or greater were considered to bediabetic. Studies were conducted 3, 7,14, 21 and28 days after the induction of diabetes. On theday of the experiment rats were anesthetizedwith Nembutal i.p. (50 mg kg-1, Abbott Labora-tories, North Chicago, IL). Following the deter-mination of motor nerve conduction velocity(MNCV) and endoneural blood flow (EBF),the abdominal aorta was isolated and occluded1-2cm above the branch of the commoniliac artery. Distal to the occlusion a solution

VASCULAR DYSFUNCTION IN DIABETIC NEUROPATHY 133

containing India ink with 2% gelatin [1,17] wasinjected to facilitate the identification of thesuperior gluteal and internal pudendal arteries,which arise from the common iliac artery. Therat was then sacrificed by exsanguination, andbody temperature lowered with topical ice.Samples of the left sciatic nerve were then takenfor determination of Na+/K+ ATPase activity,sorbitol, fructose and myo-inositol content andconjugated diene level. The lens was also col-lected for determination of glutathione levels.Levels of thiobarbituric acid reactive substances(TBARS) in the serum and lactate/pyruvateratio in the aorta were also determined. Sam-ples for blood glucose measurements were alsotaken on the day of the experiment.

Motor Nerve Conduction Velocity

MNCV was determined as previously describedusing a noninvasive procedure in the sciatic-posterior tibial conducting system in a tempera-ture controlled environment. L1,187 The left sciaticnerve was stimulated first at the sciatic notchand then at the Achilles tendon. Stimulationconsisted of single 0.2ms supra maximal (8V)pulses through a bipolar electrode (Grass $44Stimulator, Grass Medical Instruments, QuincyMA). The evoked potentials were recorded fromthe interosseous muscle with a unipolar plati-num electrode and displayed on a digital storageoscilloscope (model 54600A Hewlett Packard,Rolling Meadows, IL). MNCV was calculated bysubtracting the distal from the proximal latencymeasured in milliseconds from the stimulusartifact of the take-off of the evoked potentialand the difference was divided into the distancebetween the two stimulating electrodes meas-ured in millimeters using a Vernier caliper. TheMNCV was reported in meters per second.

Endoneural Blood Flow

Immediately after determinationsciatic endoneural nutritive blood

of MNCV,flow was

determined as described by Cameron et al. L2, 19]

The trachea was intubated for artificial ventila-tion and a carotid cannula inserted to monitormean arterial blood pressure. Core temperaturewas monitored using a rectal probe and tem-perature regulated between 36 and 37C usinga heating pad and radiant heat. The right sciaticnerve was carefully exposed by a small surgicalincision and the surrounding skin sutured to aplastic ring. The isolated area was filled withmineral oil, at 37C to a depth of I cm to minimizediffusion of hydrogen gas from the nerve.The rats were then artificially ventilated. A glassinsulated platinum microelectrode (tip 2 tm)was inserted into the sciatic nerve, proximal tothe trifurcation, and polarized at 0.25V withrespect to a reference electrode inserted subcu-taneously into the flank of the rat. Once therecording had stabilized the inspired air wasmodified to contain 10% hydrogen gas and thisgas flow continued until the hydrogen currentrecorded by the electrode had stabilized, indi-cating equilibrium of the inspired air with ar-terial blood. The hydrogen gas supply was thendiscontinued and the hydrogen clearance curverecorded until a baseline was achieved. Thehydrogen clearance data was fitted by computerto a mono- or bi-exponential curve using com-mercial software (Prism, GraphPad, San Diego,CA) and nutritive blood flow, (ml/min/100g),calculated using the equation described byYoung (1980) and vascular conductance, (ml/min/100g/mm Hg) determined by dividingnutritive blood flow by the average mean arte-rial blood pressure. Two recordings were madefor each rat at different locations along thenerve and the final blood flow value averaged.

Vascular Reactivity

Videomicroscopy was used to investigate in vitrovasodilatory responsiveness of arterioles sup-plying the region of the sciatic nerve (branchesof the superior gluteal and internal pudendalarteries) as previously described. 11 The vessels


used for these studies were generally orientedlongitudinally in relation to the sciatic nerve;however, on occasion radially oriented vesselswere also used. No differences were observed inacetylcholine-induced vasodilation based onthe orientation of the vessel to the sciatic nerve.Since vessels overlying the sciatic nerve wereused in this study they should be regarded asepineurial rather than perineurial. To isolatethese vessels the common iliac was exposedand the branch points of the internal pudendaland superior gluteal arteries identified. The ves-sels were then clamped, and tissue containingthese vessels and its branches dissected enbloc. The block of tissue was immediately sub-merged in an cooled (4C), oxygenated (20% O2,5%CO2 and 75% N2) Krebs Henseleit physio-logical saline solution (PSS) of the followingcomposition (in mM): NaC1 118, KC1 4.7,CaC12 2.5, KHaPO4 1.2, MgSO4 1.2, NaHCO3 20,Na2EDTA 0.026, and 5.5 glucose for dissection.Branches of the superior gluteal and internalpudendal arteries (50-150 tm internal diameterand 2mm in length) were carefully dissectedand trimmed of fat and connective tissue. Bothends of the isolated vessel segment were cannu-lated with glass micropipettes filled with PSS(4C), and secured with 10-0 nylon Ethilon mono-filament sutures (Ethicon, Inc., Cornelia, GA).The pipettes were attached to a single pressurereservoir (initially set at 20mmHg) under con-dition of no flow. The preparation was trans-ferred to the stage of an inverted microscope(CK2, Olympus, Lake Success, NY). Attachedto the microscope were a CCTV camera(WV-BL200, Panasonic, Secaucus, NJ), videomonitor (Panasonic), and a video caliper (VIA-100 K, Boeckeler Instruments, Inc., Tucson, AZ).The organ chamber was connected to a rotarypump (Masterflex, Cole Parmer InstrumentCo., Vernon Hills, IL), which continuously cir-culated oxygenated PSS at 30ml/min andwarmed to 37C. The pressure within the vesselwas then slowly increased to 40mmHg. At thispressure we found that KC1 gave the maximalconstrictor response. Therefore, all the studies

were conducted at 40mm Hg. Internal diameter(resolution of 2 tm) was measured by manuallyadjusting the video micrometer. After 30minequilibration, KC1 was added to the bath to testvessel viability. Vessels, which failed to con-strict more than 30%, were discarded. Afterwashing with PSS, vessels were incubatedfor 30min in PSS and then constricted withU46619 (10-8-10-7M) to 30-50% of passivediameter. There was no significant differencein the amount of U46619 required to induceconstriction in control and diabetic vessels.Cumulative concentration-response relation-ships were evaluated for acetylcholine (10-8-10-4 M) using vessels from control and diabeticrats. At the end of each acetylcholine doseresponse determination sodium nitroprusside(10-4M) was added to determine maximalvasodilation. In a separate set of experiments ar-terioles were isolated to determine superoxidelevels using hydroethidine as previouslydescribed. 20

Detection of Superoxide

Hydroethidine, an oxidative fluorescent dyewas used to evaluate in situ levels of super-oxide (O-) as described previously.I2" 21] Hydro-ethidine is permeable to cells and in the presenceof O- is oxidized to fluorescent ethidium bro-mide, where it is trapped by intercalating withDNA. This method provides sensitive detectionof O- in situ. Unfixed frozen ring segmentswere cut into 30%tm-thick sections and placedon glass slides. Hydroethidine (2x 10-6M)was topically applied to each tissue section andcoverslipped. Slides were incubated in a lightprotected humidified chamber at 37C for 30minutes. Images were obtained with a Bio-RadMRC-1024 laser scanning confocal microscopeequipped with a krypton/argon laser. Fluores-cence was detected with a 585-nm long passfilter. Normal and diabetic tissues were pro-cessed and imaged in parallel. Laser settingswere identical for acquisition of images fromnormal and diabetic specimens.


Sciatic Nerve Sorbitol, Fructoseand Myo-inositol Content

The left sciatic nerve was removed, desheathed,and weighed for determination of Na+/K+

ATPase activity and conjugated diene levels(as described below) and sorbitol, fructose andmyo-inositol content. 1"1sl For the latter deter-mination, tissue samples were boiled for10min in water containing c-D-methylmanno-pyranoside as an internal standard and depro-teinized with 0.5 ml each of 0.19M Ba(OH)2 and0.19M ZnSO4. Following centrifugation the su-pernatant was collected, frozen, and lyophilized.Afterwards, the samples were derivatized andintracellular contents determined by gas-liquidchromatography as previously described, i1,181.

Na+/K/ ATPase Activity

Total and ouabain-inhibited Na+/K+ ATPaseactivities were measured in crude homogenatesof sciatic nerve. 1"181 Sciatic nerves were de-sheathed and homogenized in a polytron, utiliz-ing 3 10 sec bursts, at 4C in I ml of 0.2M sucrose,0.02M Tris-HC1 buffer, pH 7.5. The samples werethen centrifuged at 100-x g for 10min at 4C.An aliquot of the supernatant (50 gl) was add-ed to two cuvettes containing 100mMNaC1,10mM KC1, 2.5 mMMgC12, 2mM ethyleneglycolbis(fl-aminoethyl ether)-N1-N’-tetraaceticacid (EGTA), I mM Tris-ATP, I mM 3-(cyclohex-ylammonium) phosphoenolpyruvate, 30mMimidazole-HC1 buffer (pH 7.3), 0.15mM NADH,50 gg lactate dehydrogenase, 30 gg pyruvatekinase with or without I mM ouabain to inhibitthe ouabain-sensitive Na+/K+ ATPase fraction.After a 20 min stabilization period, the oxidationof NADH was recorded over a 30min period.The activity was expressed as gmol ADP/g wetweight/h. Each assay was conducted in triplicate.

Additional Biological Parameters

Lactate/pyruvate ratios for the aorta weredetermined using perchloric acid extracts of the

tissue and high performance liquid chromato-graphy as previously described. 221 Lens glu-tathione (GSH), serum TBARS and sciatic nerveconjugated diene levels were determined as mark-ers of oxidative stress. Lens glutathione levelswere determined according to Lou et al. [23] Lenswere weighed and homogenized in cold I mlof 10% trichloroacetic acid and centrifuged for15 min at 1000- x g. The supernatant (100 gl) wasmixed with 0.89ml of 1.0M Tris, pH 8.2, and0.02M EDTA. Afterwards, 10 gl of dithionitro-benzene (DTNB) was added and change inabsorbance measured at 412nm. A glutathionestandard curve (100-500 ng) was performed foreach assay. The data were recorded gg/mg wetwt. TBARS level in serum was determined bythe method of Mihara et al. [24] as modified bySiman and Eriksson. [25] Briefly, 200 gl of serumwas boiled in 0.75 ml of phosphoric acid (0.19 M),0.25 ml thiobarbituric acid (0.42 mM) and 0.3 mlwater for 60 min. Afterwards, the samples wereprecipitated with methanol/NaOH and centri-fuged for 5 min. The supernatant was measuredfluorometrically at excitation wavelength 532 nmand emission wavelength 553nm. Standardswere prepared from malonyldialdehydbis(di-etylacetal) and were treated identically as theserum samples. The data was reported gg/mlserum. Sciatic nerve conjugated diene levelswere determined according to the method ofRecknagel and Ghoshal [26] as adapted to peri-pheral nerve tissue by Low and Nicklander. [27]

Briefly, a segment of the sciatic nerve was extract-ed with chloroform and methanol. The lipid ex-tract was evaporated and redissolved in I mlcyclohexane. Conjugated diene levels were deter-mined by measuring the absorbance at 233 nmwith extraction blanks used as references. Anextinction coefficient of 2.52 x 104M was used.

Data Analysis

The results are presented as mean +/-SE. Com-parisons between the groups (control vs. dia-betic) for MNCV, EBF, sciatic nerve Na+/K+

ATPase activity, sciatic nerve sorbitol, fructose


and myo-inositol content, serum TBARS, sciaticnerve conjugated diene, aorta lactate/pyruvateratio and lens glutathione levels were conduct-ed using independent unpaired Student’stests. Dose response curves for acetylcholine forcontrol vs. diabetic rats were compared usinga two way repeated measures analysis of vari-ance with autoregressive covariance struc-ture using proc mixed program of SAS. [1]

Whenever significant interactions were notedfor control vs. diabetic specific treatment-dose-effects were analyzed using a Bonferoni adjust-ment. A p value of less 0.05 was consideredsignificant. All computations were performedusing SAS for Windows version 6.12.

RESULTS

Body Weight and Plasma Glucose Levels

Data in Table I show that streptozotocin-induceddiabetic rats on average gained less weight thanage-matched control rats over the 3-28 dayexperimental period of this study. At the timeof experimentation plasma glucose levels wereincreased 3-4-fold in diabetic compared tocontrol rats.

Na+/K+ ATPase Activity and Sorbitol,Fructose and Myo-inositol Contentof the Sciatic Nerve

It has been reported that Na+/K+ ATPaseactivity is decreased in the sciatic nerve of

diabetic rats and this may be a contributingfactor to the slowing of motor nerve conductionvelocity. 28-31 Moreover, it has been reportedthat the sorbitol content of the sciatic nerveis increased in diabetic rats accompanied bya reciprocal decrease in the myo-inositolcontent. [18"31’32] These changes have also beenlinked to nerve dysfunction in diabeticrats. [18,31,32] In the present study we examinedthe time course for the development of thesechanges in the sciatic nerve of diabetic rats inrelation to slowing of motor nerve conductionvelocity and changes in vascular function. Datain Table II demonstrate that ouabain-sensitiveNa+/K+ ATPase activity in the sciatic nervefrom diabetic rats is decreased by about 10%following 1 week of diabetes and is signifi-cantly reduced after 4 weeks. The fructose andsorbitol content of the sciatic nerve is signifi-cantly increased after 3 and 7 days of dia-betes, respectively. In contrast, the myo-inositolcontent is significantly decreased following 1week of diabetes and after 4 weeks of diabetesis reduced by about 65%.

Lens Glutathione Level and Serum TBARS

As a measurement of oxidative stress wedetermined lens glutathione level and serumTBARS in streptozotocin-induced diabetic ratsfollowing 3- 28 days of diabetes. Data in Table IIIdemonstrate that lens glutathione levels aresignificantly decreased following 1 week ofdiabetes and serum TBARS are increased

TABLE Change in body weight and blood glucose levels

Animal Change in body weight (g/week) Blood glucose mg/dl

Control (n=11)* 35 4- 6 105 4- 8

Diabetic (Days)3 (n 7) 4 4- 4 348 4-157 (n=6) 224-6 411 +2314 (n 7) 17 4- 7 430 4- 2621 (n 7) 4 4- 4 448 4-1028 (n 7) 4- 3 458 4- 24

Data are means + S.E.M. The number of experimental observations is indicated by parenthesis.Control rats were studied at one and three weeks.p < 0.05 vs. control.


TABLE II Changes in Na+/K ATPase activity and sorbitol, fructose and myo-inositol content in the sciatic nerve ofstreptozotocin- induced diabetic rats

Ouabain-sensitive Content (nmol/mg wet wt)Na//K ATPase

Animal (tmol ADP/g wet wt/h) Sorbitol Fructose Myo-inositol

Control 235.0 4-16.4 0.2 4- 0.1 0.4 4- 0.1 11.0 4-1.4

Diabetic (Days)3 (7) 256.7 4- 31.1 0.5 4- 0.2 1.7 4- 0.2 8.7 4-1.47 (6) 209.1 4- 34.0 1.0 4- 0.3 2.7 4-1.0 6.1 4-1.214 (7) 195.54-25.0 1.04-0.1 3.1 4-0.4 5.54-1.221 (7) 193.7 4-15.0 1.1 4- 0.3 3.6 4- 0.5 5.1 4- 0.528 (7) 181.0 4-16.4 1.2 4- 0.2 3.2 4- 0.6 3.7 4- 0.4

Data are mean + S.E.M. The number of experimental observations is indicated by parenthesis.+p < 0.05 vs. control.

TABLE III Lens glutathione level and serum TBARS instreptozotocin- induced diabetic rats

Glutathione TBARSAnimal (tg/mg wet wt) (tg/ml serum)

Control 1.24 4- 0.22 11.9 4- 2.7

Diabetic (Days)

3 (7) 0.79 4- 0.13 14.7 4- 3.17 (6) 0.26 4- 0.06 14.1 4- 3.614 (7) 0.24 4- 0.05 16.7 4- 3.121 (7) 0.18 4-0.01 23.9 4- 3.228 (7) 0.28 4- 0.04 23.9 4- 3.4

Data are means 4- S.E.M. The number of experimental observations isindicated by parenthesis.p < 0.05 vs. control.

reaching significance after 3 weeks of diabetes.We also examined conjugated diene levels inthe sciatic nerve of diabetic rats. In these stud-ies levels of conjugated dienes were increasedsignificantly by about 2-3 fold as early as 1

week after the induction of diabetes (data notshown).

Motor Nerve Conduction Velocityand Endoneural Blood Flowin Diabetic Rats

Data in Table IV demonstrate that motor nerveconduction velocity was significantly decreasedafter 2 weeks of diabetes. Endoneural bloodflow reported as nutritive flow (ml/min/100 g)and conductance (ml/min/100 g/mm Hg)were significantly reduced by about 50% fol-lowing 3 days of diabetes compared to controlrats and remained reduced after 4 weeks ofdiabetes (Tab. IV). In these studies the mean ar-terial blood pressure was not significantly dif-ferent between the control and diabetic rats(data not shown).

TABLE IV Time course for the development of motor nerve conduction and endoneural blood flow defects in thestreptozotocin-induced diabetic rat

MNCV NutritiveAnimal m/sec (ml/min/100 g)

Endoneural blood flow

Conductance(ml/min/100 g/mm Hg)

Control (11) 52.4 4- 2.5 18.2 4- 2.1 0.154 4- 0.016

Diabetic (Days)3 (7) 49.6 4-1.9 9.6 4-1.5 0.084 4- 0.0127 (6) 52.6 4- 3.9 8.6 4- 2.7 0.070 4- 0.01914 (7) 41.3 4-1.7 10.64-2.4 0.097 4-0.01121 (7) 38.6 4-1.6 9.9 4-1.5 0.087 4- 0.01228 (7) 41.6 4-1.3 9.3 4-1.1 0.084 4- 0.011

Data are means 4- S.E.M. The number of experimental observations is indicated by parenthesis.+p < 0.05 vs. control.


Analysis of Superoxide Levels

Data in Figure 1 show that after 2 weeks ofdiabetes superoxide (O) levels as measured byhydroethidine fluorescence are increased inarterioles that provide circulation to the regionof the sciatic nerve compared to normal vessels.The increase in O- levels was observed in endo-thelial cells as well as in the smooth muscleand adventitial cells. Analysis of O- levels invessels from 3 and 7-day diabetic rats suggestthat the increase in O- in diabetic rats was agradual process and appeared to approachmaximum after 2 weeks of diabetes. No furtherincrease in O levels was apparent in vesselsafter 3 and 4 weeks of diabetes. As a control,preincubating vessels from diabetic rats withsuperoxide dismutase quenched the hydroethi-dine fluorescence (data not shown).

Arteriolar Vascular Reactivityin Diabetic Rats

Stimulated changes in vascular diameterwere measured in vitro by application of

acetylcholine. Ilj Baseline diameter of vesselsfrom control and diabetic rats was similar andthe vessels were constricted to a similardegree with U46619 (10 100 tM). Acetylcholineproduced a concentration-dependent vasodila-tion (endothelium-dependent) in arterioles thatprovide circulation to the region of the sciaticnerve (maximum dilation 93 4- 2%) (Fig. 2,Tab. V). At 3 days, the vascular responses toacetylcholine were similar in diabetic and nor-mal rats (Fig. 2, Tab. V). At 7 days, vascularrelaxation to acetylcholine was impaired in dia-betic rats at a lower dose of acetylcholine (1 x10-7M), however maximal relaxation was un-changed from normal animals (Fig. 2, Tab. V).After 14 days, vascular relaxation in diabetic ratswas significantly less at all doses of acetyl-choline compared ’to normal rats (Fig. 2, Tab. V).Maximal relaxation to acetylcholine was signifi-cantly impaired in vessels from 2-week diabetic(624-10%) compared to normal (934-2%) rats(Tab. V). Similar results were also obtained at3 and 4 weeks of diabetes (Tab. V). In contrast,maximal vasodilation induced by sodiumnitroprusside (endothelium-independent) was

Control Vessels

Diabetic (2 wk.) Vessels

FIGURE Detection of superoxide levels in arterioles from control and 2-week diabetic rats. Fluorescent photomicrographs ofconfocal microscopic sections of arterioles that provide circulation to the region of the sciatic nerve from three individualcontrol and 2-week streptozotocin-induced diabetic rats were examined on three separate days. Arterioles were labeled with theoxidative dye hydroethidine as described in the Methods section. Recording of fluorescent were taken at identical laser andphotomultiplier settings for both control and diabetic rats.


lOO

8o

o= 60x

rr 4o

20

I Control[] Diab. 3day

Diab. week@ Diab. 2week

8 7 6 5 4

Acetylcholine (-log M)

FIGURE 2 Acetylcholine-induced endothelium dependent relaxation of arterioles adjacent to the sciatic nerve in control anddiabetic rats. For these studies control rats and rats with diabetes for 3, 7 and 14 days were used. Pressurized arterioles wereconstricted with U46619 (30-50%) and incremental doses of acetylcholine were added to the bathing solution while recordingsteady state vessel diameter. The number of experimental animals used in these studies was the same as noted in Table I. Thedenotes that the response to acetylcholine was significantly attenuated (p < 0.05) in the diabetic rat.

TABLE V Relaxation of arterioles adjacent to the sciatic nerve from control and streptozotocin-induced diabetic rats: Effect onacetylcholine-induced EDs0 and maximum response and sodium nitroprusside maximal dilation

Acetylcholine SNPAnimal ED50(nM) Max % relaxation (% relaxation)

Control (11) 564-15 934-2 954-2

Diabetic (Days)3 (10) 71 4- 32 84 4- 6 88 4- 67 (9) 190 4- 42 89 4- 3 94 4- 314 (9) 328 4-155 62 4-10 85 4- 721 (10) 258 4-103 74 4- 5 85 4- 428 (10) 375 4-143 73 4- 8 88 4- 6

Data are means 4-S.E.M. The number of experimental observations is indicated by parenthesis./p < 0.05 vs. control.

not significantly affected by diabetes in thesevessels (Tab. V).

DISCUSSION

The results from these studies demonstratethat diabetes-induced decrease in endoneuralblood flow and impairment of acetylcholine-induced vasodilation of arterioles that provide

circulation to the region of the sciatic nerveprecedes slowing of motor nerve conductionand decrease in Na+/K+ ATPase activity inthe sciatic nerve. Moreover, our studies showthat the generation of superoxide in vascula-ture that provides circulation to the region ofthe sciatic nerve accompanies the diabetes-in-duced impairment in vasodilation. Other mark-ers of oxidative stress are also increased duringthis period in the streptozotocin-induced


diabetic rat model. These studies suggest thatvascular dysfunction may be the major factorcontributing to the development of diabeticneuropathy.

Studies by Greene and co-workershave shown that a reduction in Na+/K+ ATPaseactivity in the sciatic nerve of diabetic rats maybe linked to an increase in the sorbitol contentand reduction in myo-inositol levels in thesciatic nerve. [28-31] These disturbances havebeen proposed to be partially responsible forthe slowing of nerve conduction velocity in thediabetic rat. [18,28-32] Our studies show that thereciprocal changes in the level of sorbitol andmyo-inositol in the sciatic nerve of the diabeticrat occurs early in diabetes and precedes thereduction in Na+/K/ ATPase activity. However,in our studies slowing of motor nerve conduc-tion velocity occurs prior to the reduction inNa+/K+ ATPase activity in the sciatic nerve. Thissuggests that the early change in nerve conduc-tion velocity in the streptozotocin-induced dia-betic rat is not due to a reduction in Na+/K+

ATPase activity. It is possible that the reductionin Na+/K+ ATPase activity in the sciatic nerveenhances neural dysfunction but that the initialdefect responsible for slowing of motor nerveconduction velocity is caused by vascularchanges.Cameron et al., showed that nerve blood flow

was reduced by about 40% as early as 1 weekafter the induction of diabetes.TM In the samestudy they also showed that acute hyperglyce-mia maintained over the period required tomeasure nerve blood flow (2-4 h) caused a 50%reduction in blood flow. [2] Stevens et al., usinglaser Doppler flux to measure nerve blood flowfound that flow was variable during the. firsttwo days following streptozotocin injection,was reduced by 20% after four days of diabetesand fell steadily until reaching a plateau at 40%of control values after 4 weeks of diabetes. E331

Similar results were obtained in our studies.We found that endoneural blood flow wassignificantly reduced by about 50% three days

after the induction of diabetes and remaineddecreased for the 4-week period of the study.The determination of the sequential devel-

opment of vascular dysfunction in arteriolesthat provide circulation to the region of thesciatic nerve demonstrates that impairment ofendothelial-dependent vasodilation occurs ear-ly in diabetes. One week after the inductionof diabetes, vasodilation in response to a lowdose of acetylcholine (10-7M) was significantlydecreased. However, maximum vasodilation inresponse to acetylcholine was not significantlyreduced at this time, although two weeks afterthe induction of diabetes vasodilation in re-sponse to all doses of acetylcholine was sig-nificantly reduced and maximum vasodilationwas decreased by about 30%. This effect re-mained relatively consistent for up to 4 weeksafter the induction of diabetes. This study isamong the first to show the sequential develop-ment of diabetes-induced changes in endoneuralblood flow, vascular reactivity and motor nerveconduction velocity. It clearly demonstrates thatdiabetes-induced impairment of endothelial-dependent vasodilation by arterioles providingcirculation to the region of the sciatic nerve pre-cedes the slowing of motor nerve conductionvelocity. This study also implies that impair-ment of acetylcholine-induced vasodilation ispreceded by the reduction in endoneural bloodflow suggesting that diabetes-induced impair-ment of endothelial dependent vasodilation inthis vascular bed is not initially related to thedefect in endoneural blood flow. One possibleexplanation for this finding is that the initialreduction in endoneural blood flow is due toimpaired vasodilation of vessels within thenerve, which may occur earlier in diabetes.

Diabetes-induced impairment of vascular re-activity is a common feature in both conduitand resistance arteries from diabetic animalmodels.[34] Impaired endothelium-dependentvasodilation in conduit and resistance arteriesin patients with insulin-dependent diabeteshas also been reported. 3s’36 However, our


previous studies are among the first to showthat endothelium-dependent vasodilation byarterioles that provide circulation to the sciaticnerve is altered by diabetes. [1] The mechanismresponsible for diabetes-induced impairmentof vascular reactivity is unknown. In the aorta,diabetes causes the endothelium to increase theproduction of both superoxide and hydrogenperoxide leading to enhanced intracellular pro-duction of hydroxyl radicals. 37 Studies byPieper and colleagues have suggested that thegeneration of hydroxyl radicals may mediatethe damage caused by diabetes to the endo-thelium of the aorta. [34’37-401 Other studieshave suggested that endothelial dysfunction insmaller vessels may be mediated by the increasedproduction of free radicals and of prostaglandinendoperoxides.14 In resistance-level arteriolesof the mesenteric circulation diabetes causes areduction in endothelium-dependent vaso-dilation. [9,41] Heygate et al., reported that pre-treatment of mesenteric arteries from diabeticrats with indomethacin significantly improvedacetylcholine-induced vasodilation suggesting arole for a cyclooxygenase-derived vasoconstric-tor prostanoids in mediating diabetes-inducedvascular dysfunction in these arteries. E91 Inaddition, Rodriguez-Manas et al., reported thatpretreatment of mesenteric arteries from diabeticrats with superoxide dismutase improvedacetylcholine responsiveness suggesting thatthe increased production of superoxide anionsin the vascular wall of mesenteric arteries mayalso mediate vascular dysfunction. Elll Mayhanreported similar results using basilar arteriesfrom diabetic rats. [42] In the latter study topicalapplication of superoxide dismutase partiallyrestored acetylcholine-induced vasodilation ofthe basilar artery towards that observed innondiabetic rats suggesting that impaired vaso-dilation may be related in part to enhancedrelease of oxygen-derived free radicals.I42 Otherstudies support a role for the production ofoxygen radicals in diabetic neuropathy. Studieswith diabetic rats incorporating the use of

anti-oxidant treatment have been shown toprevent peripheral nerve dysfunction. [43-46]

In our studies we have shown that acetylcho-line-induced vasodilation of arterioles thatprovide circulation to the region of the sciaticnerve is mediated by at least two mechanismsinvolving the production of nitric oxide (NO)and endothelium-derived hyperpolarizingfactor (EDHF). 11 Therefore, impairment ofendothelium-dependent vasodilation by dia-betes in these vessels may be mediated byconditions that inhibit the production or bio-activity of either or both of these vasodilators.The present study demonstrates that diabetescauses the production/accumulation of super-oxide in these vessels. As discussed above, theproduction of oxygen radicals has been associat-ed with impairment of endothelium-dependentvasodilation in both conduit and resistancearteries. Presently, we do not know whetherthe generation of superoxide or the secondaryproduction of hydrogen peroxide or hydroxylradicals by these vessels might contribute todiabetes-induced vascular dysfunction.

In summary, these studies demonstrate thatendothelium-dependent vasodilation of arte-rioles that provide circulation to the region ofthe sciatic nerve is impaired early in diabetesand along with reduction in endoneural bloodflow precedes the slowing of motor nerveconduction velocity and reduction of sciaticnerve Na+/K+ ATPase activity. In addition thegeneration and accumulation of superoxide bythese arterioles coincides with the impairmentof vasodilation. These results suggest that vas-cular dysfunction that may be caused by theproduction of free oxygen radicals may bethe major factor contributing to the early devel-opment of diabetic neuropathy.

Acknowledgments

This work was supported by a National Instituteof Diabetes and Digestive and Kidney DiseasesGrant DK-25295, by a grant from the National


Institute of Diabetes and Digestive and KidneyDiseases DK-58005, by a Diabetes Center Grantfrom the Veterans Affairs and InternationalJuvenile Diabetes Foundation, and by a researchgrant from the American Diabetes Association.The authors would like to express their appre-ciation to Ms. Pam Tompkins for her assistancein the measurement of superoxide levels.

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